GNRH antagonists IIIB

ABSTRACT

Peptides which inhibit the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. Administration of an effective amount prevents ovulation of female mammalian eggs and/or the release of steroids by the gonads. The peptides have the structure: 
     
         X-R.sub.1 -R.sub.2 -D-Trp-Ser-R.sub.5 -R.sub.6 -Leu-Arg-Pro-R.sub.10 
    
     wherein X is hydrogen or an acyl group having 7 or less carbon atoms; R 1  is dehydro-Pro, D-pGlu, D-Phe, D-Trp or β-D-NAL; R 2  is Cl-D-Phe, F-D-Phe, C.sup.α Me-4-Cl-D-Phe, NO 2  -D-Phe, Cl 2  -D-Phe or Br-D-Phe; R 5  is Tyr, I-Tyr, CH 3  -Phe, F-Phe or Cl-Phe; R 6  is a D-isomer of a lipophilic amino acid or is 4-NH 2  -D-Phe, 4-gua-D-Phe, D-His, D-Lys, D-Orn, D-Har or D-Arg; and R 10  is Gly-NH 2 , D-Ala-NH 2  or NH-Y, with Y being lower alkyl, cycloalkyl, fluoro lower alkyl or ##STR1## where Q is H or lower alkyl. When R 1  is β-D-NAL, R 6  is 4-NH 2  -D-Phe, 4-gua-D-Phe, D-His, D-Lys, D-Orn, D-Har or D-Arg. When R 5  is Tyr or 2-Cl-Phe, either (a) Orn, AAL or aBu is substituted for Ser, or (b) R 1  is β-D-NAL and R 6  is D-His or 4-gua-D-Phe. N.sup.α Me-Leu may optionally be substituted for Leu.

This invention was made with Government support under Contract No.NO1-HD-0-2836 and/or Grant No. HD 13527 awarded by the NationalInstitutes of Health. The Government has certain rights in thisinvention.

This application is a continuation-in-part of our Ser. No. 473,876,filed Mar. 10, 1983, abandoned, and our Ser. No. 512,920, filed July 12,1983, abandoned.

The present invention relates to peptides which inhibit the release ofgonadotropins by the pituitary gland in mammalians, including humans,and to methods of preventing ovulation and/or inhibiting the release ofsteroids. More particularly, the present invention is directed topeptides which inhibit gonadal function and the release of the steroidalhormones, progesterone and testosterone.

BACKGROUND OF THE INVENTION

The pituitary gland is attached by a stalk to the region in the base ofthe brain known as the hypothalamus. In particular, follicle stimulatinghormone (FSH) and luteinizing hormone (LH), sometimes referred to asgonadotropins or gonadotropic hormones, are released by the pituitarygland. These hormones, in combination, regulate the functioning of thegonads to produce testosterone in the testes and progesterone andestrogen in the ovaries, and they also regulate the production andmaturation of gametes.

The release of a hormone by the anterior lobe of the pituitary glandusually requires a prior release of another class of hormones producedby the hypothalamus. One of the hypothalamic hormones acts as a factorthat triggers the release of the gonadotropic hormones, particularly LH,and this hormone is referred to herein as GnRH although it has also beenreferred to as LH-RH and as LRF. GnRH has been isolated andcharacterized as a decapeptide having the following structure:

    p-Glu-His-Trp-Ser-Gly-Leu-Arg-Pro-Gly-NH.sub.2

Peptides are compounds which contain two or more amino acids in whichthe carboxyl group of one acid is linked to the amino group of the otheracid. The formula for GnRH, as represented above, is in accordance withconventional representation of peptides where the amino terminus appearsto the left and the carboxyl terminus to the right. The position of theamino acid residue is identified by numbering the amino acid residuesfrom left to right. In the case of GnRH, the hydroxyl portion of thecarboxyl group of glycine has been replaced with an amino group (NH₂).The abbreviations for the individual amino acid residues above areconventional and are based on the trivial name of the amino acid, e.g.p-Glu is pyroglutamic acid, His is histidine, Trp is tryptophan, Ser isserine, Tyr is tyrosine, Gly is glycine, Leu is leucine, Orn isornithine, Arg is arginine, Pro is proline, Sar is sarcosine, Phe isphenylalanine and Ala is alanine. These amino acids together withvaline, isoleucine, threonine, lysine, aspartic acid, asparagine,glutamine, cysteine, methionine, phenylalanine, and proline aregenerally considered to be the common, naturally occurring orprotein-derived amino acids. Except for glycine, amino acids of thepeptides of the invention are of the L-configuration unless notedotherwise.

It is well known that the substitution of D-amino acids for Gly in the6-position of the GnRH decepeptide or nonapeptide provides GnRH analogshaving from substantially greater potency than GnRH to effect therelease of LH and FSH (i.e. the gonadotropins) by the pituitary gland ofmammalians. It is also known that substitution of various amino acidsfor His (or the deletion of His) at the 2-position of the GnRHdecapeptide produces analogs having an inhibitory effect on the releaseof LH and other gonadotropins by the pituitary gland of mammalians.

There are reasons for desiring to prevent ovulation in femalemammalians, and the administration of GnRH analogs that are antagonisticto the normal function of GnRH have been used to suppress or delayovulation. For this reason, analogs of GnRH which are antagonistic toGnRH are being investigated for their potential use as a contraceptiveor for regulating conception periods. GnRH antagonists may also be usedfor the treatment of precocious puberty and endometriosis to promotegrowth in female animals. Such antagonists have also been found usefulto regulate the secretion of gonadotropins in male mammals and can beemployed to arrest spermatogenesis, e.g. as male contraceptives, and fortreatment of prostatic hypertrophy. It is desired to provide improvedpeptides which are strongly antagonistic to endogenous GnRH and whichprevent secretion of LH and the release of steroids by the gonads ofmammals.

SUMMARY OF THE INVENTION

The present invention provides peptides which inhibit the release ofgonadotropins in mammalians, including humans, and it also providesmethods for inhibiting the release of steroids by the gonads of male andfemale mammalians. The improved GnRH analogs are strongly antagonisticto GnRH and have an inhibitory effect on the reproduction processes ofmammalians. These analogs may be used to inhibit the production ofgonadotropins and sex hormones under various circumstances includingprecocious puberty, hormone dependent neoplasia, dysmenorrhea andendometriosis.

Generally, in accordance with the present invention, peptides have beensynthesized which strongly inhibit the secretion of gonadotropins by thepituitary gland of mammalians, including humans, and/or inhibit therelease of steroids by the gonads. These peptides are analogs of GnRHwherein there is a 1-position substitution, such as dehydro-Pro, D-pGlu,D-Phe, D-Trp or β-(2-naphthyl)-D-alanine(hereinafter β-D-2NAL), a2-position substitution in the form of a halogenated D-Phe, a 3-positionsubstitution in the form of D-Trp or β-D-2NAL, a 6-position substitutionplus a substituent in the 5-position in the form of a halogenated L-Pheor L-Tyr and/or the substitution of a diamino acid having not more than5 carbon atoms in the 4-position. The 1-position substituent may bemodified so that its alpha amino group contains an acyl group, such asformyl, acetyl, acrylyl, vinylacetyl or benzoyl, with acetyl(Ac) andacrylyl(Acr) being preferred. Modified L-Phe or L-Tyr in the 5-positionprovides increased antagonistic activity as a result of the specificmodifications present in the phenyl ring. Single substitutions forhydrogen are made in either the 2- or 3-position, and the substitutionsare selected from chloro, fluoro, iodo, bromo and nitro, with chloro andfluoro being preferred. For example, when I-Tyr is inserted in the5-position, I is preferably present in the meta or 3-position. Whenβ-D-NAL is present in the 1-position, a hydrophillic D-amino acidresidue, such as 4-NH₂ -D-Phe, 4-guanidino-D-Phe, D-His, D-Lys, D-Orn,D-Arg or D-Har(Homoarginine) is present in the 6-position. Whendehydro-Pro is present in the 1-position, a D-isomer of a lipophilicamino acid, such as D-Trp, D-Phe, D-Leu, D-Ile, D-Nle, D-Tyr, D-Val,D-Ala, D-Ser(OtBu), β-D-2NAL or (imBzl)D-His is preferably present inthe 6-position. The substitutions in the 4-, 7- and 10-positions aregenerally considered to be optional.

Because these peptides are highly potent to inhibit release of LH, theyare often referred to as GnRH antagonists. The peptides inhibitovulation of female mammals when administered at very low levels atproestrus and are also effective to cause resorption of fertilized eggsif administered shortly after conception. These peptides are alsoeffective for the contraceptive treatment of male mammals.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

More specifically, the peptides of the present invention are representedby the following Formula I:

    X-R.sub.1 -R.sub.2 -D-Trp-R.sub.4 -R.sub.5 -R.sub.6 -R.sub.7 -Arg-Pro-R.sub.10

wherein X is hydrogen or an acyl group having 7 or less carbon atoms; R₁is dehydro-Pro, D-pGlu, D-Phe D-Trp or β-D-NAL; R₂ is Cl-D-Phe, F-D-Phe,NO₂ -D-Phe, C.sup.α Me-4-Cl-D-Phe, Cl₂ -D-Phe or Br-D-Phe; R₄ is Ser,Orn, AAL or aBu; R₅ is Tyr, 3-F-Phe, 2-F-Phe, 3-I-Tyr, 3-CH₃ -Phe, 2-CH₃-Phe, 3-Cl-Phe or 2-Cl-Phe; R₆ is a D-isomer or a lipophilic amino acidor is 4-NH₂ -D-Phe, D-Lys, D-Orn, D-Har, D-His, 4-gua-D-Phe or D-Arg; R₇is Leu or N.sup.α Me-Leu; and R₁₀ is Gly-NH₂, Gly-OCH₃, Gly-OCH₂ CH₃,Sar-NH₂, D-Ala-NH₂ or NH-Y, with Y being lower alkyl, cycloalkyl, fluorolower alkyl or ##STR2## where Q is H or lower alkyl, wherein β-D-2NALcan be substituted for D-Trp in the 3-position; provided however thatwhen R₁ is β-D-NAL, then R₆ is 4-NH₂ -D-Phe, D-Lys, D-Orn, D-Har, D-His,4-gua-D-Phe or D-Arg, and provided further that when R₅ is Tyr or2-Cl-Phe, then either (a) R₄ is Orn, AAL or aBu or (b) R₁ is β-D-2NALand R₆ is D-His or 4-gua-D-Phe.

By β-D-NAL is meant the D-isomer or alanine which is substituted bynaphthyl on the β-carbon atom, which may also be designated 3-D-NAL.Preferably β-D-2NAL is employed which means that the β-carbon atom isattached to naphthalene at the 2-position on the ring structure;however, β-D-1NAL may also be used. By AAL is meant β-amino-Ala and byaBu is meant α,γdiamino butyric acid, either of which or Orn can besubstituted for Ser in the 4-position. When such a substitution is madefor Ser, β-D-2NAL is preferably employed in the 3- and 6-positions anddehydro Pro is used in the 1-position. By 4-gua-D-Phe is meantp-guanidino-D-Phe or p-guanido-D-Phe, wherein the guanidine radical,--NHC(NH₂)═NH, is substituted in the 4- or para-position on the ring.

The peptides of the present invention can be synthesized by classicalsolution synthesis or by a solid phase technique using achloromethylated resin, a methylbenzhydrylamine resin (MBHA), abenzhydrylamine (BHA) resin or any other suitable resin known in theart. The solid phase synthesis is conducted in a manner to stepwise addthe amino acids in the chain in the manner set forth in detail in theU.S. Pat. No. 4,211,693. Side-chain protecting groups, as are well knownin the art, are preferably added to Ser, Tyr and Arg when present, aswell as to certain of the substituents, and may optionally be added toTrp, before these amino acids are coupled to the chain being built uponthe resin. Such a method provides the fully protected intermediatepeptidoresin.

The intermediates of the invention may be represented:

    X.sup.1 -R.sub.1 -R.sub.2 -D-Trp(X.sup.2)-R.sub.4 (X.sup.3)-R.sub.5 (X.sup.4)-R.sub.6 (X.sup.5)-R.sub.7 -Arg(X.sup.5)-Pro-X

wherein: X¹ is an α-amino protecting group of the type known to beuseful in the art in the stepwise synthesis of polypeptides and when Xin the desired peptide composition is a particular acyl group, thatgroup may be used as the protecting group. Among the classes of α-aminoprotecting groups covered by X¹ are (1) acyl-type protecting groups,such as formyl(For), trifluoroacetyl, phthalyl, p-toluenesulfonyl(Tos),benzoyl(Bz), benzenesulfonyl, o-nitrophenylsulfenyl(Nps),tritylsulfenyl, o-nitrophenoxyacetyl, acrylyl(Acr), chloroacetyl,acetyl(Ac) and γ-chlorobutyryl; (2) aromatic urethan-type protectinggroups, e.g., benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl,such as p-chloro-benzyloxycarbonyl(ClZ), p-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl and p-methoxybenzyloxycarbonyl; (3) aliphaticurethan protecting groups, such as tertbutyloxycarbonyl(Boc),diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl andallyloxycarbonyl; (4) cycloalkyl urethan-type protecting groups, such ascyclopentyloxycarbonyl, adamantyloxycarbonyl and cyclohexyloxycarbonyl;(5) thiourethan-type protecting groups, such as phenylthiocarbonyl; (6)alkyl-type protecting groups, such as allyl(Aly),triphenylmethyl(trityl) and benzyl(Bzl); (7) trialkylsilane groups, suchas trimethylsilane. The preferred α-amino protecting group is Boc when Xis hydrogen.

X² is hydrogen or a protecting group for the indole nitrogen, such asformyl or benzyl; however in many syntheses there is no need to protectTrp.

X³ is hydrogen or a protecting group for the alcoholic hydroxyl group ofSer, such as one selected from the group consisting of acetyl, benzoyl,tetrahydropyranyl, tert-butyl, trityl, benzyl and 2,6-dichlorobenzyl,with benzyl being preferred. Alternatively, when a substitution is madefor Ser, X³ may be a protecting group for a side chain amino group, suchas Tos, Z of ClZ.

X⁴ is hydrogen or a protecting group for the phenolic hydroxyl group ofTyr, if Tyr is present, selected from the group consisting oftetrahydropyranyl, tert-butyl, trityl, benzyl, Z,4-bromobenzyloxycarbonyl and 2,6-dichlorobenzyl. 2,6-dichlorobenzyl ispreferred.

X⁵ is a protecting group for the side chain guanidino group or the aminogroup or the imidazole group of Arg, Lys, His or the like, such asnitro, Tos, trityl, benzyloxycarbonyl, adamantyloxycarbonyl, Z, and Bocor a protecting group for Trp as X², or X⁵ may be hydrogen, which meansthere is no protection on the side chain group atoms. Tos is generallypreferred.

X⁶ may be Gly-O-CH₂ -[resin support]; O-CH₂ -[resin support];D-Ala-O-CH₂ -[resin support]; Gly-NH-[resin support] or D-Ala-NH-[resinsupport]; and it may be OH, ester, amide or hydrazide either of Gly orD-Ala or attached directly to Pro.

The criterion for selecting side chain protecting groups for X² -X⁵ isthat the protecting group should be stable to the reagent under thereaction conditions selected for removing the α-amino protecting groupat each step of the synthesis. The protecting group should not be splitoff under coupling conditions, and the protecting group should beremovable upon completion of the synthesis of the desired amino acidsequence under reaction conditions that will not alter the peptidechain.

When the X⁶ group is Gly-O-CH₂ -[resin support], D-Ala-O-CH₂ -[resinsupport] or O-CH₂ -[resin support], the ester moiety of one of the manyfunctional groups of the polystyrene resin support is being represented.When the X⁶ group is Gly-NH-[resin support] or D-Ala-NH-[resin support],an amide bond connects Gly or D-Ala to BHA resin or to a MBHA resin.

When X is acetyl, for example, in the final formula, it may be possibleto employ it as the X¹ protecting group for the α-amino group of D-NALor whatever amino acid is used in the 1-position by adding it before thecoupling of this last amino acid to the peptide chain. However, areaction is preferably carried out with the peptide on the resin (afterdeblocking the α-amino group while the side-chain groups remainprotected), e.g. by reacting with acetic acid in the presence ofdicyclohexyl carbodiimide (DCC) or preferably with acetic anhydride orby another suitable reaction as known in the art.

The fully protected peptide can be cleaved from a chloromethylated resinsupport by ammonolysis, as is well known in the art, to yield the fullyprotected amide intermediate. Deprotection of the peptide, as well ascleavage of the peptide from a benzhydrylamine resin, can take place at0° C. with hydrofluoric acid (HF). Anisole is preferably added to thepeptide prior to treatment with HF. After the removal of HF, undervacuum, the cleaved, deprotected peptide is conveniently treated withether, decanted, taken-up in dilute acetic acid and lyophilized.

Thus the invention also provides a method for making a peptide or anontoxic salt thereof, said peptide having the formula:

    X-R.sub.1 -R.sub.2 -D-Trp-R.sub.4 -R.sub.5 -R.sub.6 -R.sub.7 -Arg-Pro-R.sub.10,

wherein X is hydrogen or an acyl group having 7 or less carbon atoms; R₁is dehydro-Pro, D-pGlu, D-Phe, D-Trp or β-D-NAL; R₂ is Cl-D-Phe,F-D-Phe, NO₂ -D-Phe, C.sup.α Me-4-Cl-D-Phe, Cl₂ -D-Phe or Br-D-Phe; R₄is Ser, Orn, AAL or aBu; R₅ is Tyr, 3-F-Phe, 2-F-Phe, 3-I-Tyr, 3-CH₃-Phe, 2-CH₃ -Phe, 3-Cl-Phe or 2-Cl-Phe; R₆ is a D-isomer of a lipophilicamino acid, 4-NH₂ -D-Phe, 4-gua-D-Phe, D-His, D-Lys, D-Orn, D-Har orD-Arg; R₇ is Leu or N.sup.α Me-Leu; and R₁₀ is Gly-NH₂, Gly-OCH₃,Gly-OCH₂ CH₃, Sar-NH₂, D-Ala-NH₂ or NH-Y, with Y being lower alkyl,cycloalkyl, fluoro lower alkyl or ##STR3## where Q is H or lower alkyl,wherein β-D-2NAL can be substituted for D-Trp in the 3-position;provided however that when R₁ is β-D-NAL, then R₆ is either 4-NH₂-D-Phe, 4-gua-D-Phe, D-His, D-Lys, D-Orn or D-Arg, and provided furtherthat when R₅ is Tyr or 2-Cl-Phe, then either (a) R₄ is Orn, AAL or aBuor (b) R₁ is β-D-2NAL and R₆ is D-His or 4-gua-D-Phe; which methodcomprises (a) forming an intermediate compound having the formula:

    X.sup.1 -R.sub.1 -R.sub.2 -D-Trp(X.sup.2)-R.sub.4 (X.sup.3)-R.sub.5 (X.sup.4)-R.sub.6 (X.sup.5)-R.sub.7 -Arg(X.sup.5)-Pro-X.sup.6,

wherein X¹ is hydrogen or an α-amino protecting group; X² is hydrogen ora protecting group for the indole nitrogen; X³ is hydrogen or aprotecting group for the alcoholic hydroxyl group of Ser or for aside-chain amino group; X⁴ is hydrogen or a protecting group for thephenolic hydroxyl group of Tyr; X⁵ is hydrogen or a protecting group forthe side chain; and X⁶ is selected from the group consisting ofGly-O-CH₂ -(resin support), O-CH₂ -(resin support), D-Ala-O-CH₂ -(resinsupport), Gly-NH-(resin support), D-Ala-NH-(resin support), Gly-NH₂, andesters, amides and hydrazides; (b) splitting off one or more of thegroups X¹ to X⁵ and/or cleaving from any resin support included in X⁶and, if desired, (c) converting a resulting peptide into a nontoxic saltthereof.

Purification of the peptide is effected by ion exchange chromotographyon a CMC column, followed by partition chromotography using the elutionsystem: n-butanol; 0.1N acetic acid (1:1 volume ratio) on a columnpacked with Sephadex G-25, or by using HPLC, as known in the art.

The peptides of the invention are effective at levels of less than 100micrograms per kilogram of body weight, when administered at about noonon the day of proestrus to prevent ovulation in female rats. Forprolonged suppression of ovulation, it may be necessary to use dosagelevels in the range of from about 0.1 to about 2.5 milligrams perkilogram of body weight. These antagonists are also effective to arrestspermatogenesis when administered to male mammals on a regular basis andcan thus be used as contraceptives. Since these compounds will reducetestosterone levels (an undesired consequence in the normal, sexuallyactive male), it may be reasonable to administer replacement dosages oftestosterone along with the GnRH antagonist. These antagonists can alsobe used to regulate the production of gonadotropins and sex steroids forother purposes as indicated hereinbefore.

EXAMPLE

Peptides as indicated in TABLE I having the formula:

    Ac-R.sub.1 -4-F-D-Phe-D-Trp-Ser-R.sub.5 -R.sub.6 -Leu-Arg-Pro-R.sub.10

are prepared by the solid-phase procedure referred to above.

                                      TABLE I                                     __________________________________________________________________________    R.sub.1  R.sub.5  R.sub.6                                                                              R.sub.10                                             __________________________________________________________________________    1 β-D-2NAL                                                                        2-CH.sub.3 --Phe                                                                       D-Arg  Gly--NH.sub.2                                        2 "      3-CH.sub.3 --Phe                                                                       "      "                                                    3 "      3-I--Tyr "      "                                                    4 dehydro Pro                                                                          3-Cl--Phe                                                                              D-Trp  Gly--NH.sub.2                                        5 "      2-F--Phe "      "                                                    6 "      2-Cl--Phe (Orn.sup.4)                                                                  "      "                                                    7 "      Tyr (Orn.sup.4)                                                                        β-D-2NAL                                                                        Gly--NH.sub.2 (β-D-2NAL.sup. 3)                 8 "      2-CH.sub.3 --Phe                                                                       β-D-1NAL                                                                        Gly--NH.sub.2 (4-Cl--D-Phe.sup. 2)                   9 "      2-Cl--Phe                                                                              D-Trp  Gly--NH.sub.2                                        10                                                                              dehydro Pro                                                                          3-F--Phe D-Trp  "                                                    11                                                                              β-D-2NAL                                                                        3-F--Phe D-Arg  "                                                    12                                                                              "      Tyr      4-gua-D-Phe                                                                          "                                                    13                                                                              "      Tyr      D-His  "                                                    14                                                                              dehydro Pro                                                                          Tyr(AAL.sup.4)                                                                         D-Leu  NHCH.sub.2 CH.sub.3                                  15                                                                              D-Trp  2-F--Phe (aBu.sup.4)                                                                   D-Phe  "                                                    16                                                                              D-pGlu 2-F--Phe D-Ile  D-Ala--NH.sub.2                                      17                                                                              D-Phe  2-F--Phe (Orn.sup.4)                                                                   D-Val  Gly--NHNH.sub.2                                      __________________________________________________________________________

For purposes of an example, a representative solid phase synthesis ofPeptide No. 1 above, which is referred to as [Ac-β-D-2NAL¹, 4-F-D-Phe²,D-Trp³, 2-CH₃ -Phe⁵, D-Arg⁶ ]-GnRH is set forth hereinafter. Thispeptide has the following formula: Ac-β-D-2NAL-4-F-D-Phe-D-Trp-Ser-2-CH₃-Phe-D-Arg-Leu-Arg-Pro-Gly-NH₂

A BHA resin is used, and Boc-protected Gly is coupled to the resin overa 2-hour period in CH₂ Cl₂ using a 3-fold excess of Boc derivative andDCC as an activating reagent. The glycine residue attaches to the BHAresin by an amide bond.

Following the coupling of each amino acid residue, washing, deblockingand coupling of the next amino acid residue is carried out in accordancewith the following schedule using an automated machine and beginningwith about 5 grams of resin:

    ______________________________________                                                                        MIX                                                                           TIMES                                         STEP  REAGENTS AND OPERATIONS   MIN.                                          ______________________________________                                        1     CH.sub.2 Cl.sub.2 wash-80 ml. (2 times)                                                                 3                                             2     Methanol(MeOH) wash-30 ml. (2 times)                                                                    3                                             3     CH.sub.2 Cl.sub.2 wash-80 ml. (3 times)                                                                 3                                             4     50 percent TFA plus 5 percent 1,2-eth-                                                                  10                                                  anedithiol in CH.sub.2 Cl.sub.2 -70 ml. (2 times)                       5     Isopropyl alcohol + 1% ethanedithiol                                                                    3                                                   wash-80 ml. (2 times)                                                   6     TEA 12.5 percent in CH.sub.2 Cl.sub.2 -70 ml.                                                           5                                                   (2 times)                                                               7     MeOH wash-40 ml. (2 times)                                                                              2                                             8     CH.sub.2 Cl.sub.2 wash-80 ml. (3 times)                                                                 3                                             9     Boc-amino acid (10 mmoles) in 30 ml. of either                                                          30-300                                              DMF or CH.sub.2 Cl.sub.2, depending upon the solubility                       of the particular protected amino acid, (1 time)                              plus DCC (10 mmoles) in CH.sub.2 Cl.sub.2                               10    MeOH wash-40 ml. (2 times)                                                                              3                                             11    TEA 12.5 percent in CH.sub.2 Cl.sub.2 -70 ml.                                                           3                                                   (1 time)                                                                12    MeOH wash-30 ml. (2 times)                                                                              3                                             13    CH.sub.2 Cl.sub.2 wash-80 ml. (2 times)                                                                 3                                             ______________________________________                                    

After step 13, an aliquot may be taken for a ninhydrin test: if the testis negative, go back to step 1 for coupling of the next amino acid; ifthe test is positive or slightly positive, go back and repeat steps 9through 13.

The above schedule is used for coupling of each of the amino acids ofthe peptide of the invention after the first amino acid has beenattached. N.sup.α Boc protection is used for each of the remaining aminoacids throughout the synthesis. N.sup.α Boc-β-D-2NAL is prepared by amethod known in the art, e.g. as described in detail in U.S. Pat. No.4,234,571, issued Nov. 18, 1980. The side chain of Arg is protected withTos. OBzl is used as a side chain protecting group for the hydroxylgroup of Ser. D-Trp is left unprotected. N.sup.α Boc-β-D-2NAL isintroduced as the final amino acid. Boc-Arg(Tos) and Boc-D-Trp, whichhave low solubility in CH₂ Cl₂, are coupled using DMF:CH₂ Cl₂ mixtures.

After deblocking the α-amino group at the N-terminal, acetylation isachieved using a large excess of acetic anhydride in dichloromethane.This produces the following molecule: Ac-β-DNAL-4F-D-PheD-Trp-Ser(OBzl)-2CH₃ -Phe-D-Arg(Tos)-Leu-Arg(Tos)-Pro-Gly-NH-[resinsupport]. The cleavage of the peptide from the resin and completedeprotection of the side chains takes place very readily at 0° C. withHF. Anisole is added as a scavenger prior to HF treatment. After theremoval of HF under vacuum, the resin is extracted with 50% acetic acid,and the washings are lyophilized to provide a crude peptide powder.

Purification of the peptide is then effected by ion exchangechromatography on CMC (Whatman CM 32, using a gradient of 0.05 to 0.3MNH₄ OAc in 50/50 methanol/water) followed by partition chromatography ina gel filtration column using the elution system: n-Butanol; 0.1N Aceticacid (1:1--volume ratio).

The peptide is judged to be homogeneous using thin layer chromatographyand several different solvent systems, as well as by usingreversed-phase high pressure liquid chromatography and an aqueoustriethylammonium phosphate solution plus acetonitrile. Amino acidanalysis of the resultant, purified peptide is consistent with theformula for the prepared structure, showing substantially integer-valuesfor each amino acid in the chain. The optical rotation is measured on aphotoelectric polarimeter as [α]_(D) ²² =-27.5°±1(c=1, 50% acetic acid).

The peptide is assayed in vitro and in vivo. The in vitro test is madeusing dissociated rate pituitary cells maintained in culture for 4 daysprior to the assay. The levels of LH mediated in response to theapplication of peptides is assayed by specific radioimmunoassay for ratLH. Control dishes of cells only receive a measure which is 3 nanomolarin GnRH; experimental dishes receive a measure 3 nanomolar in GnRH plusa measure having either the present standard antagonist for comparisonpurposes i.e. [Ac-dehydro Pro¹, 4-F-D-Phe², D-Trp³,6 ]-GnRH or the testpeptide, in concentrations ranging from 0.01 to 10 nanomolar. The amountof LH secreted in the samples treated only with GnRH is compared withthat secreted by the samples treated with the peptide plus GnRH.

The ability of the test peptide to reduce the amount of LH released by 3nanomolar GnRH is compared to that of the present standard peptide. Theresults are calculated using 3-5 doses of each of the peptides by thestatistical program BIOPROG (provided by D. Rodbard NICHD) and areexpressed as a potency relative to the present standard. The standardpeptide usually blocks 50% of the LH released by GnRH at less than aratio of ##EQU1##

The peptide described hereinabove is also used to determineeffectiveness to prevent ovulation in female rats. In this test, aspecified number of mature female Sprague-Dawley rats, each having abody weight from 225 to 250 grams, is injected with 10 micrograms ofpeptide in corn oil at about noon on the day of proestrus. Proestrus isthe afternoon before estrus (ovulation). A separate female rat group isused as a control to which the peptide is not administered. Each of thecontrol female rats has ovulation at estrus; of the rats treated, noneof them ovulates. As a result, the peptide is considered to besignificantly effective to prevent ovulation of female rats at a verylow dosage, and the peptide is considered to be totally effective at adose of about ten micrograms. Additional testing is carried out at lowerdosages with the results being set forth in TABLE II hereinafter.

Peptides Nos. 2-17 are similarly sythesized and purified. After aminoacid analysis is completed, the purity is confirmed by hplc usingdifferent solvent systems. In vitro testing in similar fashion showssome of the peptides each to have a potency relative to the presentstandard antagonist as indicated. In vivo testing is carried out atvarying dosages for other of the peptides, and the results are shown inTABLE II.

                  TABLE II                                                        ______________________________________                                        Peptide     in vitro         in vivo                                          No.    [α].sub.D.sup.22                                                                 Potency*  Dose (μg)                                                                           No. Ovulating                              ______________________________________                                        1.     -27.5              1        1/18                                                                 0.5      12/19                                      2.     -29.4              1        6/18                                       3.     -24.5              5        0/10                                                                 1        2/10                                       4.     -74.9    1.02      10       9/10                                                                 5        3/10                                       5.     -84.5    1.9       10       0/10                                                                 5        2/10                                       7.     -77.5    0.29      10       0/10                                                                 2.5      1/10                                                                 1        8/10                                       9.     -79.5    1.15      5        0/10                                                                 2.5      5/10                                       10.    -73.1    0.45      10       0/6                                                                  5        2/20                                       11.    -30.2              1        6/7                                        12.    -36.6              2.5      8/10                                       13.    -31.4              2.5      4/10                                                                 1        8/16                                       ______________________________________                                         *relative to [Acdehydro Pro.sup.1, 4F--DPhe.sup. 2, DTrp.sup. 3,6 ]--GnRH                                                                              

All peptides listed in Table I are considered effective to blockGnRH-induced LH secretion in vitro at some reasonable concentration.Many of these peptides are much more potent in vivo than the presentstandard.

All of the peptides are considered to be effective to prevent ovulationof female mammals at very low dosages, and some selected ones areconsidered to be at least twice as potent as any GnRH antagonistspreviously known and tested.

The peptides of the invention are often administered in the form ofpharmaceutically acceptable, nontoxic salts, such as acid additionsalts, or of metal complexes, e.g., with zinc, barium, calcium,magnesium, aluminum or the like (which are considered as addition saltsfor purposes of this application), or of combinations of the two.Illustrative of such acid addition salts are hydrochloride,hydrobromide, sulphate, phosphate, nitrate, oxalate, fumarate,gluconate, tannate, maleate, acetate, citrate, benzoate, succinate,alginate, malate, ascorbate, tartrate and the like. If the activeingredient is to be administered in tablet form, the tablet may containa pharmaceutically-acceptable diluent which includes a binder, such astragacanth, corn starch or gelatin; a disintegrating agent, such asalginic acid; and a lubricant, such as magnesium stearate. Ifadministration in liquid form is desired, sweetening and/or flavoringmay be used as part of the pharmaceutically-acceptable diluent, andintravenous administration in isotonic saline, phosphate buffersolutions or the like may be effected.

The pharmaceutical compositions will usually contain the peptide inconjunction with a conventional, pharmaceutically-acceptable carrier.Usually, the dosage will be from about 1 to about 100 micrograms of thepeptide per kilogram of the body weight of the host when givenintravenously; oral dosages will be higher. Overall, treatment ofsubjects with these peptides is generally carried out in the same manneras the clinical treatment using other antagonists of GnRH.

These peptides can be administered to mammals intravenously,subcutaneously, intramuscularly, orally, percutaneously, e.g.intranasally or intravaginally to achieve fertility inhibition and/orcontrol and also in applications calling for reversible suppression ofgonadal activity, such as for the management of precocious puberty orduring radiation-or chemo-therapy. Effective dosages will vary with theform of administration and the particular species of mammal beingtreated. An example of one typical dosage form is a bacteriostatic watersolution containing the peptide which solution is administered toprovide a dose in the range of about 0.1 to 2.5 mg/kg of body weight.Oral administration of the peptide may be given in either solid form orliquid form.

Although the invention has been described with regard to its preferredembodiments, it should be understood that changes and modifications aswould be obvious to one having the ordinary skill in this art may bemade without departing from the scope of the invention which is setforth in the claims which are appended hereto. For example, othersubstitutions known in the art which do not significantly detract fromthe effectiveness of the peptides may be employed in the peptides of theinvention. The substitutions in the phenyl ring of the Phe² residue mayalso be in the 3-position and in the 2,4 positions, which are consideredequivalents. Changes in the positions of the substituents in the residuein the 5-position may also be made.

Particular features of the invention are emphasized in the claims whichfollow.

What is claimed is:
 1. A peptide or a pharmaceutically acceptablenontoxic salt thereof, said peptide having the formula:

    X-R.sub.1 -R.sub.2 -R.sub.3 -R.sub.4 -R.sub.5 -R.sub.6 -R.sub.7 -Arg-Pro-R.sub.10

wherein X is hydrogen or an acyl group having 7 or less carbon atoms; R₁is dehydro-Pro, D-pGlu, D-Phe, D-Trp, β-D-2NAL or β-D-1NAL; R₂ is4-Cl-D-Phe, 4-F-D-Phe, 4-NO₂ -D-Phe, C.sup.α Me-4Cl-D-Phe, 3,4-Cl₂-D-Phe or 4-Br-D-Phe; R₃ is D-Trp or β-D-2NAL; R₄ is Ser, Orn, AAL oraBu; R₅ is Tyr, 3-F-Phe, 2-F-Phe, 3-I-Tyr, 3-CH₃ -Phe, 2-CH₃ -Phe,3-Cl-Phe or 2-Cl-Phe; R₆ is D-Trp, D-Phe, D-Leu, D-Ile, D-Nle, D-Tyr,D-Val, D-Ala, D-Ser(OtBu), β-D-2NAL, (imBzl)D-His, 4-NH₂ -D-Phe, D-Lys,D-Orn, D-Har, D-His, 4-guanido-D-Phe or D-Arg; R₇ is Leu or N.sup.αMe-Leu; and R₁₀ is Gly-NH₂, Gly-OCH₃, Gly-OCH₂ CH₃, Sar-NH₂, D-Ala-NH₂or NH-Y, with Y being lower alkyl, fluoro lower alkyl or NH-CONH-Q,where Q is H or lower alkyl; provided however that when R₁ is β-D-NAL,R₆ is 4-NH₂ -D-Phe, D-Lys, D-Orn, D-Har, D-His, 4-guanido-D-Phe orD-Arg, and provided further that when R₅ is Tyr or 2-Cl-Phe, then either(a) R₄ is Orn, AAL or aBu or (b) R₁ is β-D-2NAL and R₆ is D-His or4-guanido-D-Phe.
 2. A peptide in accordance with claim 1 wherein R₂ is4-Cl-D-Phe or 4-F-D-Phe.
 3. A peptide in accordance with claim 2 whereinR₁ is β-D-2NAL and R₆ is D-Arg.
 4. A peptide in accordance with claim 3wherein R₅ is 2-CH₃ -Phe, 3-CH₃ -Phe or 3-I-Tyr and R₄ is Ser.
 5. Apeptide in accordance with claim 4 wherein X is acetyl or acrylyl and R₇is Leu.
 6. A peptide in accordance with claim 2 wherein X is acetyl, R₁is dehydro-Pro, R₄ is Ser, R₅ is 3-F-Phe, R₆ is D-Trp and R₇ is Leu. 7.A peptide in accordance with claim 1 wherein X is acetyl, R₁ isβ-D-2NAL, R₂ is 4-F-D-Phe, R₄ is Ser, R₅ is 2-CH₃ -Phe, 3-I-Tyr or 3-CH₃-Phe, and R₆ is D-Arg.
 8. A peptide in accordance with claim 7 whereinR₅ is 2-CH₃ -Phe.
 9. A peptide in accordance with claim 8 wherein R₃ isD-Trp, R₇ is Leu and R₁₀ is Gly-NH₂.
 10. A peptide in accordance withclaim 7 wherein R₅ is 3-I-Tyr.
 11. A peptide in accordance with claim 10wherein R₃ is D-Trp, R₇ is Leu and R₁₀ is Gly-NH₂.
 12. A peptide inaccordance with claim 2 wherein X is acetyl and R₁ is dehydro-Pro.
 13. Apeptide in accordance with claim 12 wherein R₆ is D-His.
 14. A peptidein accordance with claim 13 wherein R₅ is Tyr.
 15. A peptide inaccordance with claim 14 wherein R₂ is 4-F-D-Phe, R₃ is D-Trp, R₄ isSer, R₇ is Leu and R₁₀ is Gly-NH₂.
 16. A peptide in accordance withclaim 1 wherein R₄ is Orn, AAL or ABu.
 17. A peptide in accordance withclaim 16 wherein R₁ is dehydro Pro and R₃ and R₆ are β-D-2NAL.
 18. Apeptide in accordance with claim 1 having the formulaacetyldehydro-Pro-4-F-D-Phe-β-D-2NAL-Orn-Tyr-β-D-2NAL-Leu-Arg-Pro-Gly-NH₂.19. A pharmaceutical composition for regulating the secretion ofgonadotropins in mammals comprising as an active ingredient an effectiveamount of a peptide as defined in claim 1 in association with a majoramount of a nontoxic diluent.
 20. A method for regulating the secretionof gonadotropins comprising administering an effective amount of apeptide or a pharmaceutically acceptable nontoxic salt thereof, saidpeptide having the formula: X-R₁ -R₂ -R₃ -R₄ -R₅ -R₆ -R₇ -Arg-Pro-R₁₀wherein X is hydrogen or an acyl group having 7 or less carbon atoms; R₁is dehydro-Pro, D-pGlu, D-Phe, D-Trp, β-D-2NAL or β-D-1NAL; R₂ is4-Cl-D-Phe, 4-F-D-Phe, 4-NO₂ -D-Phe, C.sup.α Me-4Cl-D-Phe, 3,4-Cl₂-D-Phe or 4-Br-D-Phe; R₃ is D-Trp or β-D-2NAL; R₄ is Ser, Orn, AAL oraBu; R₅ is Tyr, 3-F-Phe, 2-F-Phe, 3-I-Tyr, 3-CH₃ -Phe, 2-CH₃ -Phe,3-Cl-Phe or 2-Cl-Phe; R₆ is D-Trp, D-Phe, D-Leu, D-Ile, D-Nle, D-Tyr,D-Val, D-Ala, D-Ser(OtBu), β-D-2NAL, (imBzl)D-His, 4-NH₂ -D-Phe, D-Lys,D-Orn, D-Har, D-His, 4-guanido-D-Phe or D-Arg; R₇ is Leu or N.sup. αMe-Leu; and R₁₀ is Gly-NH₂, Gly-OCH₃, Gly-OCH₂ CH₃, Sar-NH₂, D-Ala-NH₂or NH-Y, with Y being lower alkyl, fluoro lower alkyl or NH-CONH-Q,where Q is H or lower alkyl; provided however that when R₁ is β-D-NAL,R₆ is 4-NH₂ -D-Phe, D-Lys, D-Orn, D-Har, D-His, 4-guanido-D-Phe orD-Arg, and provided further that when R₅ is Tyr or 2-Cl-Phe, then either(a) R₄ is Orn, AAL or aBu or (b) R₁ is β-D-2NAL and R₆ is D-His or4-guanido-D-Phe.
 21. A peptide or a pharmaceutically acceptable nontoxicsalt thereof, said peptide having the formula: X-R₁ -R₂ -R₃ -R₄ -R₅ -R₆-R₇ -Arg-Pro-R₁₀ wherein X is hydrogen or an acyl group having 7 or lesscarbon atoms; R₁ is dehydro-Pro, D-pGlu, D-Phe, D-Trp, β-D-2NAL orβ-D-1NAL; R₂ is 4-Cl-D-Phe, 4-F-D-Phe, 4-NO₂ -D-Phe, C.sup.αMe-4Cl-D-Phe, 3,4-Cl₂ -D-Phe or 4-Br-D-Phe; R₃ is D-Trp or β-D-2NAL; R₄is Orn, AAL or aBu; R₅ is Tyr, 3-F-Phe, 2-F-Phe, 3-I-Tyr, 3-CH₃ -Phe,2-CH₃ -Phe, 3-Cl-Phe or 2-Cl-Phe; R₆ is D-Trp, D-Phe, D-Leu, D-Ile,D-Nle, D-Tyr, D-Val, D-Ala, D-Ser(OtBu), β-D-2NAL, (imBzl)D-His, 4-NH₂-D-Phe, D-Lys, D-Orn, D-Har, D-His, 4-guanido-D-Phe or D-Arg; R₇ is Leuor N.sup.α Me-Leu; and R₁₀ is Gly-NH₂, Gly-OCH₃, Gly-OCH.sub. 2 CH₃,Sar-NH₂, D-Ala-NH₂ or NH-Y, with Y being lower alkyl, fluoro lower alkylor NHCONH-Q, where Q is H or lower alkyl; provided however that when R₁is β-D-NAL, R₆ is 4-NH₂ -D-Phe, D-Lys, D-Orn, D-Har, D-His,4-guanido-D-Phe or D-Arg.